Overview

  • Product name
    Anti-KPNB1 antibody [3E9]
    See all KPNB1 primary antibodies
  • Description
    Mouse monoclonal [3E9] to KPNB1
  • Host species
    Mouse
  • Tested applications
    Suitable for: Flow Cyt, ChIP, IP, Inhibition Assay, WB, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Dog, Human, Pig, African green monkey, Syrian hamster
  • Immunogen

    Full length native protein (purified) corresponding to Cow KPNB1. Purified from Bovine erythrocytes.

  • Positive control
    • WB: MDBK cell lysate ICC: MDBK cells, PTK cells
  • General notes

    Previously labelled as NTF97/Importin beta. 

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer
    Preservative: 0.05% Sodium azide
    Constituent: PBS
  • Concentration information loading...
  • Purity
    Protein A purified
  • Primary antibody notes
    The accumulation of proteins in the nucleus is mediated by short sequences of basic amino acids called nuclear localization sequences (NLSs). These sequences are necessary and sufficient to direct a protein or inert carrier to the nuclear interior. Nuclear protein import is accomplished by two sequential, energy dependent events. The first step, docking at the nuclear pore complex, requires a 54/56 kDa nuclear localization signal receptor (a-karyopherin, importin-a, SRP-a) and the nuclear transport factor p97 (NTF 97, b-karyopherin, importin-b). The second step, translocation across the nuclear envelope (NE), requires the GTP-binding protein, Ran/TC4.
  • Clonality
    Monoclonal
  • Clone number
    3E9
  • Isotype
    IgG2a
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab2811 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Flow Cyt 1/100.

ab170191 - Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.

ChIP Use at an assay dependent concentration.
IP Use at an assay dependent concentration.
Inhibition Assay Use at an assay dependent concentration.
WB 1/5000. Detects a band of approximately 97 kDa (predicted molecular weight: 97 kDa).
ICC/IF 1/1000.

Target

  • Function
    Functions in nuclear protein import, either in association with an adapter protein, like an importin-alpha subunit, which binds to nuclear localization signals (NLS) in cargo substrates, or by acting as autonomous nuclear transport receptor. Acting autonomously, serves itself as NLS receptor. Docking of the importin/substrate complex to the nuclear pore complex (NPC) is mediated by KPNB1 through binding to nucleoporin FxFG repeats and the complex is subsequently translocated through the pore by an energy requiring, Ran-dependent mechanism. At the nucleoplasmic side of the NPC, Ran binds to importin-beta and the three components separate and importin-alpha and -beta are re-exported from the nucleus to the cytoplasm where GTP hydrolysis releases Ran from importin. The directionality of nuclear import is thought to be conferred by an asymmetric distribution of the GTP- and GDP-bound forms of Ran between the cytoplasm and nucleus. Mediates autonomously the nuclear import of ribosomal proteins RPL23A, RPS7 and RPL5. Binds to a beta-like import receptor binding (BIB) domain of RPL23A. In association with IPO7 mediates the nuclear import of H1 histone. In vitro, mediates nuclear import of H2A, H2B, H3 and H4 histones. In case of HIV-1 infection, binds and mediates the nuclear import of HIV-1 Rev. Imports PRKCI into the nucleus.
  • Sequence similarities
    Belongs to the importin beta family.
    Contains 8 HEAT repeats.
    Contains 1 importin N-terminal domain.
  • Cellular localization
    Cytoplasm. Nucleus envelope.
  • Information by UniProt
  • Database links
  • Alternative names
    • IMB 1 antibody
    • IMB1 antibody
    • IMB1_HUMAN antibody
    • Impnb antibody
    • Importin 90 antibody
    • Importin beta 1 antibody
    • Importin beta 1 subunit antibody
    • Importin subunit beta-1 antibody
    • Importin-90 antibody
    • IPOB antibody
    • Karyopherin beta 1 antibody
    • Karyopherin beta 1 subunit antibody
    • Karyopherin subunit beta-1 antibody
    • KPNB 1 antibody
    • Kpnb1 antibody
    • MGC2155 antibody
    • MGC2156 antibody
    • MGC2157 antibody
    • NTF 97 antibody
    • NTF97 antibody
    • NTF97/Importin beta antibody
    • Nuclear factor P97 antibody
    • Pore targeting complex 97 kDa subunit antibody
    • PTAC97 antibody
    see all

Images

  • All lanes : Anti-KPNB1 antibody [3E9] (ab2811)

    Lane 1 : U251 cell lysate
    Lane 2 : HepG2 cell lysate
    Lane 3 : C6 cell lysate

    Lysates/proteins at 25 µg per lane.

    Predicted band size: 97 kDa
    Observed band size: 100 kDa
    why is the actual band size different from the predicted?

  • Immunofluorecent analysis of HMVEC (Human Lung Microvascular Endothelial cells) cells stained for KPNB1 using ab2811.

  • NIH3T3 cells were incubated for 4 minutes in PHEM/1%triton, washed for 2 minutes in 1x PHEM and fixed for 10 minutes at room temperature in 3.7% PFA containing 30mM sucrose. Following washing in PBS, the cells were incubated for 2 minutes in 100% Methanol at -20°C, then washed 3 times in PBS. The cells were then incubated with ab2811 (1/200) for 1 hour at room temperature. The image panel shows the nuclei stained with DAPI (blue) and the nuclear envalope and cytoplasm stained with ab2811 (green). 100x magnification.

  • Immunoprecipitation of Importin beta, in HeLa cells, using ab2811. Coomassie-stained 8% SDS-page gel was loaded with IP fractions obtained by incubating 2 mg of pre-cleared HeLa whole cell extracts with 4µg ab2811 or 4µg IgG (control). The Importin band (see arrow) was cut out of the gel and its identity confirmed by Mass Spectometry. Please refer to protocol tab for further experimental details.
  • Flow cytometry analysis of KPNB1 in Jurkat cells (green) compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/ml, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with ab2811 (1ug/test) for 40 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated secondary antibody and re-suspended in PBS for FACS analysis.

  • All lanes : Anti-KPNB1 antibody [3E9] (ab2811) at 1/2000 dilution

    Lane 1 : Mouse lung whole tissue lysate
    Lane 2 : Mouse kidney whole tissue lysate
    Lane 3 : Mouse spleen whole tissue lysate

    Secondary
    All lanes : HRP-conjugated Goat anti-mouse IgG polyclonal at 1/5000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 97 kDa
    Observed band size: 97 kDa


    Exposure time: 3 minutes

    See Abreview

  • Anti-KPNB1 antibody [3E9] (ab2811) at 1/5000 dilution + HeLa Whole cell extract

    Secondary
    HRP-conjugated anti mouse IgG at 1/5000 dilution

    Performed under reducing conditions.

    Predicted band size: 97 kDa



    HeLa whole cell extract was run on a 10%SDS-PAGE and transferred to PVDF membrane. Membrane was blocked for 30 mins in TBS/0.1% Tween/ 5% Milk; ab2811 (1/5000) was incubated for 1 hr in TBS/0.1%Tween/5% Milk and followed by 3 washes in TBS/ 0.1%Tween (3x 7 mins). Secondary antibody was incubated for 30 mins in a TBS/ 0.1% Tween solution.This was followed by 3 washes with the TBST solution (3x7 mins) and one wash in TBS. Western blot developed using ECL plus (Amersham).
  • Immunolocalization of KPNB1 in PTK cells using ab2811.

  • Flow cytometry analysis of KPNB1 in PC12 cells (green) compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/ml, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with ab2811 (1ug/test) for 40 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated secondary antibody and re-suspended in PBS for FACS analysis.

  • Flow cytometry analysis of KPNB1 in 3T3 cells (green) compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/ml, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with ab2811 (1ug/test) for 40 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated secondary antibody and re-suspended in PBS for FACS analysis.

  • Overlay histogram showing Jurkat cells stained with ab2811 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab2811, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Jurkat cells fixed with methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

References

This product has been referenced in:
  • Agostini-Dreyer A  et al. IGFBP-3 Induced by Ribotoxic Stress Traffics From the Endoplasmic Reticulum to the Nucleus in Mammary Epithelial Cells. J Endocr Soc 3:517-536 (2019). Read more (PubMed: 30788454) »
  • de Castro IJ  et al. CDK1 and PLK1 coordinate the disassembly and reassembly of the nuclear envelope in vertebrate mitosis. Oncotarget 9:7763-7773 (2018). Read more (PubMed: 29487689) »
See all 35 Publications for this product

Customer reviews and Q&As

21-22 of 22 Abreviews or Q&A

Answer

Thank you for your inquiry and I am sorry to hear that you are having trouble with ab2811. I did notice that you had sent the same image on your two complaints. For ab2811, you said that you didn't see any staining in immunocytochemistry (ICC) with rat bone marrow mesenchymal stem cells fixed with PFA for 1 week. For ab8295, you said that you didn't see any staining in PFA perfusion fixed tissues (IHC) with rat heart, however both complaints showed the same image (attached). Can you please explain this discrepancy? We would recommend not fixing for 1 week as this may overfix the epitope. Also since this protein is part of the nuclear envelope, we would strongly recommend permeabilization. We recommend to try this tested protocol for ICC with ab2811: NIH3T3 cells were incubated for 4 minutes in PHEM/1%triton, washed for 2 minutes in 1x PHEM and fixed for 10 minutes at room temperature in 3.7% PFA containing 30mM sucrose. Following washing in PBS, the cells were incubated for 2 minutes in 100% Methanol at -20°C, then washed 3 times in PBS. The cells were then incubated with ab2811 (1/200) for 1 hour at room temperature. The image panel shows the nuclei stained with DAPI (blue) and the nuclear envalope and cytoplasm stained with ab2811 (green). 100x magnification. https://www.abcam.com/NTF97-Importin-beta-antibody-3E9-ab2811.html#ab2811_2.jpg You can also see the ICC protocol tested with ab2811 in this reference: http://www.ncbi.nlm.nih.gov/pubmed/21832065?dopt=Abstract I hope this information helps. Please contact us with any other questions.

Read More
Application
Western blot
Sample
Cow Cell lysate - nuclear (mammary epithelial cells)
Gel Running Conditions
Reduced Denaturing (8%)
Loading amount
30 µg
Specification
mammary epithelial cells
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

Ms. Allyson Dreyer

Verified customer

Submitted Jul 29 2011

21-22 of 22 Abreviews or Q&A

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